Efficacy of a broad host range lytic bacteriophage against E. coli adhered to urothelium

Persistent urinary tract infections (UTI) are often caused by E. coli adhered to urothelium. This type of cells is generally recognized as very tolerant to antibiotics which renders difficult the treatment of chronic UTI. This work investigates the use of lytic bacteriophages as alternative antimicrobial agents, particularly the interaction of phages with E. coli adhered to urothelium and specifically determines their efficiency against this type of cells. The bacterial adhesion to urothelium was performed varying the bacterial cell concentrations and the period and conditions (static, shaken) of adhesion. Three collection bacteriophages (T1, T4 and phiX174 like phages) were tested against clinical E. coli isolates and only one was selected for further infection experiments. Based on the lytic spectrum against clinical isolates and its ability to infect the highest number of antibiotic resistant strains, the T1-like bacteriophage was selected. This bacteriophage caused nearly a 45 % reduction of the bacterial population after 2 h of treatment. This study provides evidence that bacteriophages are effective in controlling suspended and adhered cells and therefore can be a viable alternative to antibiotics to control urothelium adhered bacteria

Genetically manipulated phages with improved pH resistance for oral administration in veterinary medicine

Orally administered phages to control zoonotic pathogens face important challenges, mainly related to the hostile conditions found in the gastrointestinal tract (GIT). These include temperature, salinity and primarily pH, which is exceptionally low in certain compartments. Phage survival under these conditions can be jeopardized and undermine treatment. Strategies like encapsulation have been attempted with relative success, but are typically complex and require several optimization steps. Here we report a simple and efficient alternative, consisting in the genetic engineering of phages to display lipids on their surfaces. Escherichia coli phage T7 was used as a model and the E. coli PhoE signal peptide was genetically fused to its major capsid protein (10A), enabling phospholipid attachment to the phage capsid. The presence of phospholipids on the mutant phages was confirmed by High Performance Thin Layer Chromatography, Dynamic Light Scattering and phospholipase assays. The stability of phages was analysed in simulated GIT conditions, demonstrating improved stability of the mutant phages with survival rates 102107 pfu.mL1 higher than wild-type phages. Our work demonstrates that phage engineering can be a good strategy to improve phage tolerance to GIT conditions, having promising application for oral administration in veterinary medicine.This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and under the scope of the Project PTDC/BBB-BSS/6471/2014 (POCI-01-0145-FEDER-016678). Franklin L. Nobrega and Ana Rita Costa acknowledge FCT for grants SFRH/BD/86462/2012 and SFRH/BPD/94648/2013, respectively. Melvin F. Siliakus acknowledges funding from the Biobased Ecologically Balanced Sustainable Industrial Chemistry (BE-BASIC) foundation. Electron microscopy work was performed at the Wageningen Electron Microscopy Centre (WEMC) of Wageningen University

The effect of the timing of exposure to Campylobacter jejuni on the gut microbiome and inflammatory responses of broiler chickens

Background Campylobacters are an unwelcome member of the poultry gut microbiota in terms of food safety. The objective of this study was to compare the microbiota, inflammatory responses, and zootechnical parameters of broiler chickens not exposed to Campylobacter jejuni with those exposed either early at 6 days old or at the age commercial broiler chicken flocks are frequently observed to become colonized at 20 days old. Results Birds infected with Campylobacter at 20 days became cecal colonized within 2 days of exposure, whereas birds infected at 6 days of age did not show complete colonization of the sample cohort until 9 days post-infection. All birds sampled thereafter were colonized until the end of the study at 35 days (mean 6.1 log10 CFU per g of cecal contents). The cecal microbiota of birds infected with Campylobacter were significantly different to age-matched non-infected controls at 2 days post-infection but generally the composition of the cecal microbiota were more affected by bird age as the time post infection increased. The effects of Campylobacter colonization on the cecal microbiota were associated with reductions in the relative abundance of OTUs within the taxonomic family Lactobacillaceae and the Clostridium cluster XIVa. Specific members of the Lachnospiraceae and Ruminococcaceae families exhibit transient shifts in microbial community populations dependent upon the age at which the birds become colonized by C. jejuni. Analysis of ileal and cecal chemokine/cytokine gene expression revealed increases in IL-6, IL-17A and Il-17F consistent with a Th17 response but the persistence of the response was dependent on the stage/time of C. jejuni colonization that coincide with significant reductions in the abundance of Clostridium cluster XIVa. Conclusions This study combines microbiome data, cytokine/chemokine gene expression with intestinal villus and crypt measurements to compare chickens colonized early or late in the rearing cycle to provide insights into the process and outcomes of Campylobacter colonization. Early colonization results in a transient growth rate reduction and pro-inflammatory response but persistent modification of the cecal microbiota. Late colonization produces pro-inflammatory responses with changes in the cecal microbiota that will endure in market ready chickens

Phage therapy is effective against infection by Mycobacterium ulcerans in a murine footpad model

Author Summary: Buruli Ulcer (BU), caused by Mycobacterium ulcerans, is a necrotizing disease of the skin, subcutaneous tissue and bone. Standard treatment of BU patients consists of a combination of the antibiotics rifampicin and streptomycin for 8 weeks. However, in advanced stages of the disease, surgical resection of the destroyed skin is still required. The use of bacterial viruses (bacteriophages) for the control of bacterial infections has been considered as an alternative or a supplement to antibiotic chemotherapy. By using a mouse model of M. ulcerans footpad infection, we show that mice treated with a single subcutaneous injection of the mycobacteriophage D29 present decreased footpad pathology associated with a reduction of the bacterial burden. In addition, D29 treatment induced increased levels of IFN-γ and TNF in M. ulcerans -infected footpads, correlating with a predominance of a mononuclear infiltrate. These findings suggest the potential use of phage therapy in BU, as a novel therapeutic approach against this disease, particularly in advanced stages where bacteria are found primarily in an extracellular location in the subcutaneous tissue, and thus immediately accessible by lytic phages.This work was supported by a grant from the Health Services of Fundacao Calouste Gulbenkian, and the Portuguese Science and Technology Foundation (FCT) fellowships SFRH/BPD/64032/2009, SFRH/BD/41598/2007, and SFRH/BPD/68547/2010 to GT, TGM, and AGF, respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Orally Administered P22 Phage Tailspike Protein Reduces Salmonella Colonization in Chickens: Prospects of a Novel Therapy against Bacterial Infections

One of the major causes of morbidity and mortality in man and economically important animals is bacterial infections of the gastrointestinal (GI) tract. The emergence of difficult-to-treat infections, primarily caused by antibiotic resistant bacteria, demands for alternatives to antibiotic therapy. Currently, one of the emerging therapeutic alternatives is the use of lytic bacteriophages. In an effort to exploit the target specificity and therapeutic potential of bacteriophages, we examined the utility of bacteriophage tailspike proteins (Tsps). Among the best-characterized Tsps is that from the Podoviridae P22 bacteriophage, which recognizes the lipopolysaccharides of Salmonella enterica serovar Typhimurium. In this study, we utilized a truncated, functionally equivalent version of the P22 tailspike protein, P22sTsp, as a prototype to demonstrate the therapeutic potential of Tsps in the GI tract of chickens. Bacterial agglutination assays showed that P22sTsp was capable of agglutinating S. Typhimurium at levels similar to antibodies and incubating the Tsp with chicken GI fluids showed no proteolytic activity against the Tsp. Testing P22sTsp against the three major GI proteases showed that P22sTsp was resistant to trypsin and partially to chymotrypsin, but sensitive to pepsin. However, in formulated form for oral administration, P22sTsp was resistant to all three proteases. When administered orally to chickens, P22sTsp significantly reduced Salmonella colonization in the gut and its further penetration into internal organs. In in vitro assays, P22sTsp effectively retarded Salmonella motility, a factor implicated in bacterial colonization and invasion, suggesting that the in vivo decolonization ability of P22sTsp may, at least in part, be due to its ability to interfere with motility… Our findings show promise in terms of opening novel Tsp-based oral therapeutic approaches against bacterial infections in production animals and potentially in humans

Phage engineering: how advances in molecular biology and synthetic biology are being utilized to enhance the therapeutic potential of bacteriophages

Background The therapeutic potential of bacteriophages has been debated since their first isolation and characterisation in the early 20th century. However, a lack of consistency in application and observed efficacy during their early use meant that upon the discovery of antibiotic compounds research in the field of phage therapy quickly slowed. The rise of antibiotic resistance in bacteria and improvements in our abilities to modify and manipulate DNA, especially in the context of small viral genomes, has led to a recent resurgence of interest in utilising phage as antimicrobial therapeutics. Results In this article a number of results from the literature that have aimed to address key issues regarding the utility and efficacy of phage as antimicrobial therapeutics utilising molecular biology and synthetic biology approaches will be introduced and discussed, giving a general view of the recent progress in the field. Conclusions Advances in molecular biology and synthetic biology have enabled rapid progress in the field of phage engineering, with this article highlighting a number of promising strategies developed to optimise phages for the treatment of bacterial disease. Whilst many of the same issues that have historically limited the use of phages as therapeutics still exist, these modifications, or combinations thereof, may form a basis upon which future advances can be built. A focus on rigorous in vivo testing and investment in clinical trials for promising candidate phages may be required for the field to truly mature, but there is renewed hope that the potential benefits of phage therapy may finally be realised

Bacteriophage Therapy To Reduce Campylobacter jejuni Colonization of Broiler Chickens

Colonization of broiler chickens by the enteric pathogen Campylobacter jejuni is widespread and difficult to prevent. Bacteriophage therapy is one possible means by which this colonization could be controlled, thus limiting the entry of campylobacters into the human food chain. Prior to evaluating the efficacy of phage therapy, experimental models of Campylobacter colonization of broiler chickens were established by using low-passage C. jejuni isolates HPC5 and GIIC8 from United Kingdom broiler flocks. The screening of 53 lytic bacteriophage isolates against a panel of 50 Campylobacter isolates from broiler chickens and 80 strains isolated after human infection identified two phage candidates with broad host lysis. These phages, CP8 and CP34, were orally administered in antacid suspension, at different dosages, to 25-day-old broiler chickens experimentally colonized with the C. jejuni broiler isolates. Phage treatment of C. jejuni-colonized birds resulted in Campylobacter counts falling between 0.5 and 5 log(10) CFU/g of cecal contents compared to untreated controls over a 5-day period postadministration. These reductions were dependent on the phage-Campylobacter combination, the dose of phage applied, and the time elapsed after administration. Campylobacters resistant to bacteriophage infection were recovered from phage-treated chickens at a frequency of <4%. These resistant types were compromised in their ability to colonize experimental chickens and rapidly reverted to a phage-sensitive phenotype in vivo. The selection of appropriate phage and their dose optimization are key elements for the success of phage therapy to reduce campylobacters in broiler chickens